Clustered Wavelength Division Light Detection Systems And Methods of Using The Same

ABSTRACT

Systems for detecting light (e.g., in a flow stream) are described. Light detection systems according to certain embodiments include a wavelength separator configured to generate first, second and third predetermined spectral ranges of light from a light source and first, second and third light detection modules configured to receive each of the first, second and third predetermined spectral ranges of light, the light detection modules having a plurality of photodetectors and an optical component that conveys light having a predetermined sub-spectral range to the photodetectors. Systems and methods for measuring light emitted by a sample (e.g., in a flow stream) and kits having three or more wavelength separators, a plurality of photodetectors and an optical component are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. § 119(e), this application claims priority to thefiling dates of U.S. Provisional patent application Ser. No. 62/971,840filed Feb. 7, 2020; the disclosure of which applications is incorporatedherein by reference.

INTRODUCTION

Light detection is often used to characterize components of a sample(e.g., biological samples), for example when the sample is used in thediagnosis of a disease or medical condition. When a sample isirradiated, light can be scattered by the sample, transmitted throughthe sample as well as emitted by the sample (e.g., by fluorescence).Variations in the sample components, such as morphologies, absorptivityand the presence of fluorescent labels may cause variations in the lightthat is scattered, transmitted or emitted by the sample. Thesevariations can be used for characterizing and identifying the presenceof components in the sample. To quantify these variations, the light iscollected and directed to the surface of a detector. The amount of lightthat reaches the detector can impact the overall quality of the opticalsignal outputted by the detector. The amount of light that reaches thedetector can be raised by increasing the surface area of the detector orby increasing collection of the light from the sample.

One technique that utilizes light detection to characterize thecomponents in a sample is flow cytometry. Using data generated from thedetected light, distributions of the components can be recorded andwhere desired material may be sorted. A flow cytometer typicallyincludes a sample reservoir for receiving a fluid sample, such as ablood sample, and a sheath reservoir containing a sheath fluid. The flowcytometer transports the particles (including cells) in the fluid sampleas a cell stream to a flow cell, while also directing the sheath fluidto the flow cell. Within the flow cell, a liquid sheath is formed aroundthe cell stream to impart a substantially uniform velocity on the cellstream. The flow cell hydrodynamically focuses the cells within thestream to pass through the center of a light source in a flow cell.Light from the light source can be detected as scatter or bytransmission spectroscopy or can be absorbed by one or more componentsin the sample and re-emitted as luminescence.

SUMMARY

Aspects of the present disclosure include clustered wavelength divisionlight detection systems having three or more wavelength separators thatpass light having a predetermined spectral range. The present disclosureprovides light detection systems which separate detected light intospectral ranges and require fewer reflections of the light in order togenerate a plurality of sub-spectral ranges detected by photodetectorsin the subject systems. The inventors have discovered that reflectionsto generate distinct spectral ranges of light result in increasing lightloss, in certain instances causing poor detector signal quality (e.g.,low signal to noise ratio). The present disclosure reduces the amount oflight loss that result from reflections by optical components ingenerating distinct spectral ranges for light detection. According tocertain embodiments, as described in greater detail below, the presentdisclosure is capable of generating or more distinct spectral ranges oflight while exhibiting a light loss of 20% or less, such as 19% or less,such as 18% or less, such as 17% or less, such as 16% or less, such as15% or less and including generating 20 or more distinct spectral rangesof light while exhibiting a light loss of 10% or less. In someembodiments, light detection systems are configured to generate 2 ormore distinct spectral ranges of light for every reflection by anoptical component (e.g., dichroic mirror), such as 3 or more distinctspectral ranges. In certain instances, the light detection system isconfigured to generate 30 distinct spectral ranges of light from 10reflections by optical components or less, such as generating 30distinct spectral ranges of light from 9 reflections by opticalcomponents or less.

Light detection systems according to certain embodiments include awavelength separator configured to generate first, second and thirdpredetermined spectral ranges of light from a light source and first,second and third light detection modules configured to receive each ofthe first, second and third predetermined spectral ranges of light, thelight detection modules having a plurality of photodetectors and anoptical component that conveys light having a predetermined sub-spectralrange to the photodetectors. In certain instances, the wavelengthseparator is a prism or a diffraction grating. In certain embodiments,light detection systems include three or more wavelength separators thatare each configured to pass light having a predetermined spectral rangeand one or more light detection modules in optical communication witheach wavelength separator having a plurality of photodetectors and anoptical component that conveys light having a predetermined sub-spectralrange to the photodetectors. In some embodiments, the wavelengthseparators are configured to convey light between each other. Thewavelength separators may be positioned along a single plane or alongtwo or more parallel planes. In certain embodiments, the wavelengthseparators are positioned in a polygonal configuration, such as apentagonal or hexagonal configuration. In embodiments, the wavelengthseparators are configured to pass light of a predetermined spectralrange. In some embodiments, the wavelength separators are configured topass light having wavelengths that range from 200 nm to 1200 nm, such asfrom 360 nm to 960 nm. In some embodiments, the wavelength separatorsare each configured to pass light having a spectral range that spansfrom 75 nm to 150 nm. In certain instances, the wavelength separatorsare each configured to pass light having a spectral range that spans 100nm (e.g., pass light having wavelengths that range from 360 nm to 460nm).

Light detection systems include one or more light detection modules inoptical communication with each wavelength separator. In embodiments,each light detection module includes a plurality of photodetectors andan optical component configured to convey light having a predeterminedsub-spectral range to the photodetectors. In some embodiments, eachoptical component is configured to pass light having a sub-spectralrange of from 5 nm to 50 nm to each photodetector, such as asub-spectral range of about 20 nm to each photodetector. Thephotodetectors and optical components may be positioned in each lightdetection module along a single plane or along two or more parallelplanes. In certain embodiments, the photodetectors and opticalcomponents are positioned in a polygonal configuration, such as ahexagonal, heptagonal or octagonal configuration in each light detectionmodule.

Aspects of the present disclosure also include systems for measuringlight from a sample (e.g., in a flow stream). In certain embodiments,systems include a light source and a clustered wavelength division lightdetection system that include three or more wavelength separators thatare each configured to pass light having a predetermined spectral rangeand one or more light detection modules in optical communication witheach wavelength separator having a plurality of photodetectors and anoptical component that conveys light having a predetermined sub-spectralrange to the photodetectors. In some embodiments, systems also includean optical collection system for propagating light to the lightdetection system. The optical collection system may be a free-spacelight relay system or may include fiber optics such as a fiber opticslight relay bundle. In some embodiments, the system is a flow cytometer.

Aspects of the disclosure also include methods for irradiating a sample(e.g., in a flow stream) in an interrogation field with a light source,collecting and detecting light from the sample with the subject lightdetection systems and measuring the detected light at one or morewavelengths. In some embodiments, light is collected and conveyed to thelight detection system by a free-space light relay system. In otherembodiments, light is collected and conveyed to the light detectionsystem by fiber optics, such as a fiber optics light relay bundle.

Kits including one or more components of the subject light detectionsystems are also provided. Kits according to certain embodiments,include three or more wavelength separators, a plurality ofphotodetectors and an optical component. In embodiments, the opticalcomponent includes a collimator, beam splitter, a wavelength separatoror a combination thereof. Kits may also include one or morephotodetectors, such as photomultiplier tubes (e.g., metal packagephotomultiplier tubes).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a configuration of wavelength separators positionedalong two parallel planes in a light detection system according tocertain embodiments.

FIG. 1B depicts a configuration of wavelength separators positioned in apolygonal configuration in a light detection system according to certainembodiments.

FIG. 2 depicts a wavelength separator that is configured to generatefirst, second and third spectral ranges of light according to certainembodiments.

FIG. 3 depicts components of a light detection module positioned alongtwo parallel axes according to certain embodiments.

FIG. 4 depicts components of a light detection module positioned in apolygonal configuration according to certain embodiments.

FIG. 5 depicts components of a light detection module positioned alongtwo parallel planes according to certain embodiments.

FIG. 6 depicts a light detection system having a plurality of wavelengthseparators and light detection modules according to certain embodiments.

FIG. 7A-7E depicts light detection systems having 3 or moreconcentrically arranged wavelength separators optically coupled to lightdetection modules according to certain embodiments. FIG. 7A depicts alight detection system having three wavelength separators opticallycoupled to light detection modules. FIG. 7B depicts a light detectionsystem having four wavelength separators optically coupled to lightdetection modules. FIG. 7C depicts a light detection system having fivewavelength separators optically coupled to light detection modules. FIG.7D depicts a light detection system having six wavelength separatorsoptically coupled to light detection modules. FIG. 7E depicts a threedimensional view of the light detection system of FIG. 7D.

DETAILED DESCRIPTION

Systems for detecting light (e.g., in a flow stream) are described.Light detection systems according to certain embodiments include awavelength separator configured to generate first, second and thirdpredetermined spectral ranges of light from a light source and first,second and third light detection modules configured to receive each ofthe first, second and third predetermined spectral ranges of light, thelight detection modules having a plurality of photodetectors and anoptical component that conveys light having a predetermined sub-spectralrange to the photodetectors. Systems and methods for measuring lightemitted by a sample (e.g., in a flow stream) and kits having three ormore wavelength separators, a plurality of photodetectors and an opticalcomponent are also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

Light Detections Systems

Aspects of the present disclosure include clustered wavelength divisionlight detection systems configured for detecting light from a sample(e.g., light obtained from a flow stream of a flow cytometer). Lightdetection systems according to certain embodiments include a wavelengthseparator configured to generate first, second and third predeterminedspectral ranges of light from a light source and first, second and thirdlight detection modules configured to receive each of the first, secondand third predetermined spectral ranges of light, the light detectionmodules having a plurality of photodetectors and an optical componentthat conveys light having a predetermined sub-spectral range to thephotodetectors. In certain instances, the wavelength separator is aprism or a diffraction grating. In some embodiments, light detectionsystems include three or more wavelength separators that are eachconfigured to pass light having a predetermined spectral range and oneor more light detection modules in optical communication with eachwavelength separator having a plurality of photodetectors and an opticalcomponent that conveys light having a predetermined sub-spectral rangeto the photodetectors.

In embodiments, light from a sample is divided into three or morespectral ranges by passing the light through the one or more wavelengthseparators. Each spectral range of light generated by the wavelengthseparators is further divided into smaller sub-spectral ranges which aredetected by the photodetectors. In some embodiments, light detected fromthe sample is emitted light such as fluorescent light. In otherembodiments, light detected from the sample is scattered light. The term“scattered light” is used herein in its conventional sense to refer tothe propagation of light energy from particles in the sample (e.g.,flowing in a flow stream) that are deflected from the incident beampath, such as by reflection, refraction or deflection of the beam oflight.

In embodiments, light detection systems as described herein areconfigured to exhibit little to no light loss from the light collectedfrom the sample. In some embodiments, light loss due to conveyance oflight through the subject light detection system is 25% or less, such as20% or less, such as 15% or less, such as 10% or less, such as 5% orless, such as 1% or less, such as 0.5% or less, such as 0.1% or less,such as 0.01% or less and including 0.001% or less. In certaininstances, there is no light loss from propagating light from the samplethrough the subject light detection systems (i.e., shows no measureablelight loss). For example, the amount of light from the sample decreasesby 1 mW/cm 2 or less when conveyed through the subject light detectionsystems, such as 0.5 mW/cm 2 or less, such as 0.1 mW/cm 2 or less, suchas 0.05 mW/cm 2 or less, such as 0.01 mW/cm 2 or less, such as 0.005mW/cm 2 or less, such as 0.001 mW/cm 2 or less, such as 0.0005 mW/cm 2or less, such as 0.0001 mW/cm 2 or less, such as 0.00005 mW/cm 2 or lessand including 0.00001 mW/cm 2 or less.

As described herein, light detection systems are configured to generatea plurality of sub-spectral ranges of light from the light collectedfrom the sample. In some embodiments, 5 or more sub-spectral ranges oflight are generated from the light collected from the sample, such as 10or more, such as 15 or more, such as 20 or more, such as 25 or more,such as 30 or more, such as 35 or more, such as 40 or more, such as 45or more and including 50 or more sub-spectral ranges of light. In theseembodiments, the light loss exhibited by light detections systems andmethods described herein is 20% or less, such as 19% or less, such as18% or less, such as 17% or less, such as 16% or less, such as 15% orless and including exhibiting a light loss of 10% or less. For example,5 or more sub-spectral ranges of light may be generated from the lightcollected from the sample where the light from the sample decreases by 1mW/cm 2 or less when conveyed through the subject light detectionsystems, such as 0.5 mW/cm 2 or less, such as 0.1 mW/cm 2 or less, suchas 0.05 mW/cm 2 or less, such as 0.01 mW/cm 2 or less, such as 0.005mW/cm 2 or less, such as 0.001 mW/cm 2 or less, such as 0.0005 mW/cm 2or less, such as 0.0001 mW/cm 2 or less, such as mW/cm 2 or less andincluding 0.00001 mW/cm 2 or less.

Light propagating through the subject light detection system exhibitslittle to no divergence. In other words, there is little, if any, changeto the light beam as it conveys through the wavelength separators and tothe photodetectors. In some embodiments, the focal radius of lightconveyed through the subject light detection systems increases by 5% orless, such as 4% or less, such as 3% or less, such as 2% or less, suchas 1% or less, such as 0.5% or less, such as or less, such as 0.01% orless, such as 0.001% or less and including 0.0001% or less. In certaininstances, the focal radius of light conveyed through the subject lightdetection systems does not increase at all (i.e., shows no measureableincrease in focal radius) For example, depending on the size of thelight beam conveyed through the light detection system, the diameter ofthe beam of light increases by 2 mm or less, such as 1.5 mm or less,such as 1 mm or less, such as 0.9 mm or less, such as 0.8 mm or less,such as 0.7 mm or less, such as 0.6 mm or less, such as 0.5 mm or less,such as 0.4 mm or less, such as mm or less, such as 0.2 mm or less, suchas 0.1 mm or less, such as 0.05 mm or less, such as 0.01 mm or less,such as 0.001 mm or less, such as 0.0001 mm or less and including0.00001 mm or less. In certain instances, the diameter of the beam oflight exhibits no measurable increase when conveyed through the lightdetection system (i.e., increases by 0 mm).

In some embodiments, wavelength separators are configured to generatethree or more predetermined spectral ranges of light from a light source(e.g., light from a sample irradiated with light, as described in detailbelow), such as 4 or more, such as 5 or more, such as 6 or more, such as7 or more, such as 8 or more, such as 9 or more, such as 10 or more,such as 15 or more, such as 25 or more, such as 50 or more, such as 75or more and including 100 or more predetermined spectral ranges oflight. In certain instances, light detection systems include awavelength separator configured to generate first, second and thirdpredetermined spectral ranges of light from a light source.

In some embodiments, light detection systems include 3 or morewavelength separators, such as 4 or more, such as 5 or more, such as 6or more, such as 7 or more, such as 8 or more, such as 9 or more, suchas 10 or more, such as 15 or more, such as 25 or more, such as 50 ormore, such as 75 or more and including 100 or more wavelengthseparators. The term “wavelength separator” is used herein in itsconventional sense to refer to an optical component that is configuredto separate light collected from the sample into predetermined spectralranges. In some embodiments, the wavelength separator is configured toseparate light collected from the sample into predetermined spectralranges by passing light having a predetermined spectral range andreflecting one or more remaining spectral ranges of light. In otherembodiments, the wavelength separator is configured to separate lightcollected from the sample into predetermined spectral ranges by passinglight having a predetermined spectral range and absorbing one or moreremaining spectral ranges of light. In yet other embodiments, thewavelength separator is configured to spatially diffract light collectedfrom the sample into predetermined spectral ranges. Each wavelengthseparator may be any convenient light separation protocol, such as oneor more dichroic mirrors, bandpass filters, diffraction gratings, beamsplitters or prisms. In some embodiments, the wavelength separator is aprism. In other embodiments, the wavelength separator is a diffractiongrating. In certain embodiments, wavelength separators in the subjectlight detection systems are dichroic mirrors.

In embodiments, the wavelength separators are configured to pass lighthaving wavelengths that range from a first wavelength, X_(i) (innanometers, nm) to a second wavelength X_(n) (in nanometers, nm). Insome embodiments, the wavelength separators are configured to pass lighthaving wavelengths that range from X_(i) to X_(n), such as from 100 nmto 1500 nm, such as from 150 nm to 1450 nm, such as from 200 nm to 1400nm, such as from 250 nm to 1350 nm, such as from 300 nm to 1300 nm, suchas from 350 nm to 1250 nm, such as from 400 nm to 1200 nm, such as from450 nm to 1150 nm, such as from 500 nm to 1100 nm, such as from 550 nmto 1050 nm and including passing light having wavelengths that rangefrom 600 nm to 1000 nm. In certain embodiments, wavelength separators inlight detection systems of interest are configured to pass light havingwavelengths that range from 360 nm to 960 nm.

In embodiments, wavelength separators of interest are each configured togenerate predetermined spectral ranges of light, X_(s) (in nanometers,nm). The predetermined spectral ranges may vary, where in certainembodiments, wavelength separators of interest are configured togenerate spectral ranges (X_(s)) of light that span from 50 nm to 300nm, such as from 75 nm to 275 nm, such as from 100 nm to 250 nm, such asfrom 125 nm to 225 nm and including from 150 nm to 200 nm. In certainembodiments, each wavelength separator is configured to generate aspectral range of light that spans 100 nm (i.e., X_(s)=100 nm).

In one example, light detection systems include a wavelength separatorthat is configured to generate a first predetermined spectral range oflight of from 360 nm to 480 nm; a second predetermined spectral range oflight of from 480 nm to 600 nm; a third predetermined spectral range oflight of from 600 nm to 720 nm; a fourth predetermined spectral range oflight of from 720 nm to 840 nm; and a fifth predetermined spectral rangeof light of from 840 nm to 960 nm.

In another example, light detection systems include a first wavelengthseparator configured to pass light having a wavelength that ranges from360 nm to 480 nm (i.e., X_(s)=120 nm); a second wavelength separatorconfigured to pass light having a wavelength that ranges from 480 nm to600 nm; a third wavelength separator configured to pass light having awavelength that ranges from 600 nm to 720 nm; a fourth wavelengthseparator configured to pass light having a wavelength that ranges from720 nm to 840 nm; and a fifth wavelength separator configured to passlight having a wavelength that ranges from 840 nm to 960 nm.

In some embodiments, light detection systems of interest include threeor more wavelength separators that are in optical communication witheach other, such as being positioned to convey light between each other.The wavelength separators may be oriented with respect to each other inthe light detection system (as referenced in an X-Z plane) at an angleranging from 10° to 180°, such as from 15° to 170°, such as from 20° to160°, such as from 25° to 150°, such as from 30° to 120° and includingfrom 45° to 90°. In some instances, the wavelength separators arepositioned along a single plane. In other instances, the wavelengthseparators are positioned along more than one plane. For example, thewavelength separators may be positioned along two or more parallelplanes, such as three or more, such as four or more and including fiveor more parallel planes. In certain instances, the wavelength separatorsare arranged into a geometric configuration, where arrangements ofinterest include, but are not limited to a square configuration,rectangular configuration, trapezoidal configuration, triangularconfiguration, hexagonal configuration, heptagonal configuration,octagonal configuration, nonagonal configuration, decagonalconfiguration, dodecagonal configuration, circular configuration, ovalconfiguration as well as irregular shaped configurations. In certainembodiments, the wavelength separators are arranged in a pentagonalconfiguration. In other embodiments, the wavelength separators arearranged in a hexagonal configuration.

In some embodiments, the wavelength separators are configured to conveylight between each other. In some instances, each wavelength separatoris configured to pass a spectral range of light and to convey (e.g., byreflection) one or more remaining spectral ranges of light to anotherwavelength separator. In one example, the light detection systemincludes 3 wavelength separators. The first wavelength separator isconfigured to receive light from the sample and to pass a first spectralrange of light and convey a second spectral range of light to the secondwavelength separator. The second wavelength separator is configured topass a third spectral range of light and to convey a fourth spectralrange of light to the third wavelength separator. In some instances, thethird spectral range of light is a portion of the second spectral rangeof light, such as a spectral range that spans 90% or less of the secondspectral range of light, such 85% or less, such as 80% or less, such as75% or less, such as 70% or less, such as 65% or less, such as 60% orless, such as 55% or less, such as 50%. The third wavelength separatoris configured to pass a fifth spectral range of light. In someinstances, the fifth spectral range of light is a portion of the fourthspectral range of light, such as a spectral range that spans 90% or lessof the fourth spectral range of light, such 85% or less, such as 80% orless, such as 75% or less, such as 70% or less, such as 65% or less,such as 60% or less, such as 55% or less, such as 50%.

In another example, the light detection system includes 5 wavelengthseparators. The first wavelength separator is configured to receivelight from the sample and to pass a first spectral range of light andconvey a second spectral range of light to the second wavelengthseparator. The second wavelength separator is configured to pass a thirdspectral range of light and to convey a fourth spectral range of lightto the third wavelength separator. In some instances, the third spectralrange of light is a portion of the second spectral range of light, suchas a spectral range that spans 90% or less of the second spectral rangeof light, such 85% or less, such as 80% or less, such as 75% or less,such as 70% or less, such as 65% or less, such as 60% or less, such as55% or less, such as 50%. The third wavelength separator is configuredto pass a fifth spectral range of light and to convey a sixth spectralrange of light to the fourth wavelength separator. In some instances,the fifth spectral range of light is a portion of the fourth spectralrange of light, such as a spectral range that spans 90% or less of thefourth spectral range of light, such 85% or less, such as 80% or less,such as 75% or less, such as 70% or less, such as 65% or less, such as60% or less, such as 55% or less, such as 50%. The fourth wavelengthseparator is configured to pass a seventh spectral range of light and toconvey an eighth spectral range of light to the fifth wavelengthseparator. In some instances, the seventh spectral range of light is aportion of the sixth spectral range of light, such as a spectral rangethat spans 90% or less of the sixth spectral range of light, such 85% orless, such as 80% or less, such as 75% or less, such as 70% or less,such as 65% or less, such as 60% or less, such as 55% or less, such as50%. The fifth wavelength separator is configured to pass a ninthspectral range of light. In some instances, the ninth spectral range oflight is a portion of the eighth spectral range of light, such as aspectral range that spans 90% or less of the eighth spectral range oflight, such 85% or less, such as 80% or less, such as 75% or less, suchas 70% or less, such as 65% or less, such as 60% or less, such as 55% orless, such as 50%.

In certain embodiments, the light detection system includes 5 wavelengthseparators configured to separate light having wavelengths ranging from360 nm to 960 nm, where the first wavelength separator is configured topass light having a wavelength ranging from 360 nm to 480 nm and toconvey light having a wavelength that ranges from 480 nm to 960 nm tothe second wavelength separator; the second wavelength separator isconfigured to pass light having a wavelength ranging from 480 nm to 600nm and to convey light having a wavelength that ranges from 600 nm to960 nm to the third wavelength separator; the third wavelength separatoris configured to pass light having a wavelength ranging from 600 nm to720 nm and to convey light having a wavelength that ranges from 720 nmto 960 nm to the fourth wavelength separator; and the fourth wavelengthseparator is configured to pass light having a wavelength ranging from720 nm to 840 nm and to convey light having a wavelength ranging from840 nm to 960 nm to the fifth wavelength separator. In this embodiment,the fifth wavelength separator is configured to pass light having awavelength ranging from 840 nm to 960 nm.

FIG. 1A depicts a configuration of wavelength separators positionedalong two parallel planes in a light detection system according tocertain embodiments. Light from a sample is conveyed through an opticalcollection system OC having an optical component configured to passlight having a set of wavelengths 101 (e.g., 360 nm to 960 nm) to afirst wavelength separator WS1 that is configured to pass a firstspectral range of light SR1 (e.g., 360 nm to 480 nm) and convey lighthaving a set of wavelengths 102 (e.g., 480 nm to 960 nm) to a secondwavelength separator WS2. The second wavelength separator WS2 isconfigured to pass a second spectral range of light SR2 (e.g., 480 nm to600 nm) and convey light having a set of wavelengths 103 (e.g., 600 nmto 960 nm) to a third wavelength separator WS3. The third wavelengthseparator WS3 is configured to pass a third spectral range of light SR3(e.g., 600 nm to 720 nm) and convey light having a set of wavelengths104 (e.g., 720 nm to 960 nm) to a fourth wavelength separator WS4. Thefourth wavelength separator WS4 is configured to pass a fourth spectralrange of light SR4 (e.g., 720 nm to 840 nm) and convey light having afifth spectral range of light SR5 (e.g., 840 nm to 960 nm). In thisembodiment, light is conveyed along a zig-zag light path. Each ofspectral ranges of light SR1, SR2, SR3, SR4 and SR5 are conveyed to oneor more light detection modules (as described in greater detail below).

FIG. 1B depicts a configuration of wavelength separators positioned in apolygonal configuration in a light detection system according to certainembodiments. Light from a sample is conveyed through an opticalcollection system OC having an optical component configured to passlight having a set of wavelengths 201 (e.g., 200 nm to 1200 nm) to afirst wavelength separator WS1 that is configured to pass a firstspectral range of light SR1 (e.g., 200 nm to 400 nm) and convey lighthaving a set of wavelengths 202 (e.g., 400 nm to 1200 nm) to a secondwavelength separator WS2. The second wavelength separator WS2 isconfigured to pass a second spectral range of light SR2 (e.g., 400 nm to600 nm) and convey light having a set of wavelengths 203 (e.g., 600 nmto 1200 nm) to a third wavelength separator WS3. The third wavelengthseparator WS3 is configured to pass a third spectral range of light SR3(e.g., 600 nm to 800 nm) and convey light having a set of wavelengths204 (e.g., 800 nm to 1200 nm) to a fourth wavelength separator WS4. Thefourth wavelength separator WS4 is configured to pass a fourth spectralrange of light SR4 (e.g., 800 nm to 1000 nm) and convey light having afifth spectral range of light SR5 (e.g., 1000 nm to 1200 nm). In thisembodiment, light is conveyed along a star-shaped light path. Each ofspectral ranges of light SR1, SR2, SR3, SR4 and SR5 are conveyed to oneor more light detection modules (as described in greater detail below).

FIG. 2 depicts a wavelength separator that is configured to generatefirst, second and third spectral ranges of light according to certainembodiments. Light from a sample is conveyed through an opticalcollection system OC having an optical component configured to passlight having a set of wavelengths (e.g., 200 nm to 1200 nm) to awavelength separator WS that is configured to generate a first spectralrange of light SR1, a second spectral range of light SR2 and a thirdspectral range of light SR3. Each of SR1, SR2, SR3 are each conveyed toa first, second and third light detection module (as described ingreater detail below).

As summarized above, light detection systems include one or more lightdetection modules that are configured to receive the predeterminedspectral ranges of light generated by the one or more wavelengthseparators. In some embodiments, light detection systems include awavelength separator configured to generate first, second and thirdpredetermined spectral ranges of light and first, second and third lightdetection modules configured to receive each of the first, second andthird predetermined spectral ranges of light.

In some embodiments, light detection systems a light detection module inoptical communication with each wavelength separator. In someembodiments, the light detection modules are positioned in physicalcontact with the wavelength separator, such as where an opening to thelight detection module is physically coupled to the wavelengthseparator. In other embodiments, each light detection module ispositioned from the wavelength separator by 0.001 mm or more, such as by0.005 mm or more, such as by 0.01 mm or more, such as by 0.05 mm ormore, such as by 0.1 mm or more, such as by 0.5 mm or more, such as by 1mm or more, such as by 2 mm or more, such as by 3 mm or more, such as by4 mm or more, such as by 5 mm or more, such as by 10 mm or more, such asby 15 mm or more, such as by 25 mm or more and including by 50 mm ormore. For instance, each light detection module may be positioned fromthe wavelength separator by a distance of from 0.0001 mm to 100 mm, suchas from 0.0005 mm to 95 mm, such as from 0.001 mm to 90 mm, such as from0.005 mm to 85 mm, such as from 0.01 mm to 80 mm, such as from 0.05 mmto 75 mm, such as from 0.1 mm to 70 mm, such as from 0.5 mm to 65 mm,such as from 1 mm to 60 mm, such as from 1.5 mm to 55 mm and includingfrom 2 mm to 50 mm.

Light detection modules may be releasably connected to the wavelengthseparator. The term “releasably” is used herein in its conventionalsense such that each light detection module or wavelength separator maybe freely detached and re-attached. Light detection modules orwavelength separators may be connected by any convenient protocol. Incertain embodiments, the light detection modules and wavelengthseparators are connected together with a fastener, such as a hook andloop fasteners, magnets, latches, notches, countersinks, counter-bores,grooves, pins, tethers, hinges, Velcro, non-permanent adhesives or acombination thereof. In certain instances, a light detection module isconnected to a wavelength separator by slot-fitting the wavelengthseparator into a groove of the light detection module. In yet otherinstances, a wavelength separator is connected to a light detectionmodule by one or more screws.

In some embodiments, light from each wavelength separator is conveyed toeach light detection module by an optical collection system. Eachoptical collection system may be any suitable light collection protocolthat collects the spectral range of light passed by the wavelengthseparator and directs the light to the light detection module. In someembodiments, the optical collection system includes fiber optics, suchas a fiber optics light relay bundle. In other embodiments, the opticalcollection system is a free-space light relay system.

In embodiments, each optical collection system may be physically coupledto the light detection module, such as with an adhesive, co-moldedtogether or integrated into each light detection module. In certainembodiments, each light detection module and optical collection systemare integrated into a single unit. In some instances, each lightdetection module is coupled to an optical collection system with anconnector that fastens the optical collection system to each lightdetection module, such as with a hook and loop fasteners, magnets,latches, notches, countersinks, counter-bores, grooves, pins, tethers,hinges, Velcro, non-permanent adhesives or a combination thereof.

In other embodiments, each light detection module and optical collectionsystem are in optical communication, but are not physically in contact.In embodiments, the optical collection system may be positioned 0.001 mmor more from the light detection module, such as 0.005 mm or more, suchas 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more,such as 0.5 mm or more, such as 1 mm or more, such as 10 mm or more,such as 25 mm or more, such as 50 mm or more and including 100 mm ormore from the light detection module.

In certain embodiments, the optical collection system includes fiberoptics. For example, the optical collection system may be a fiber opticslight relay bundle and the spectral range of light passed by thewavelength separator is conveyed through the fiber optics light relaybundle to the light detection module. Any fiber optics light relaysystem may be employed to convey light, where in certain embodiments,suitable fiber optics light relay systems include, but are not limitedto, fiber optics light relay systems such as those described in U.S.Pat. No. 6,809,804, the disclosure of which is herein incorporated byreference.

In other embodiments, each optical collection system is a free-spacelight relay system. The phrase “free-space light relay” is used hereinin its conventional sense to refer to light propagation that employs aconfiguration of one or more optical components to direct the spectralrange of light passed by the wavelength separator to the light detectionmodule through free-space. In certain embodiments, the free-space lightrelay system includes a housing having a proximal end and a distal end,the proximal end being coupled to the light detection module. Thefree-space relay system may include any combination of different opticalcomponents, such as one or more of lenses, mirrors, slits, pinholes,wavelength separators, or a combination thereof. For example, in someembodiments, free-space light relay systems of interest include one ormore focusing lens. In other embodiments, the subject free-space lightrelay systems include one or more mirrors. In yet other embodiments, thefree-space light relay system includes a collimating lens. In certainembodiments, suitable free-space light relay systems for propagating thespectral range of light from a wavelength separator include, but are notlimited to, light relay systems such as those described in U.S. Pat.Nos. 7,643,142; 7,728,974 and 8,223,445, the disclosures of which isherein incorporated by reference.

The light detection modules may be arranged (e.g., co-mounted together)in any geometric configuration in the subject light detection systems asdesired. The light detection modules may be arranged along one or moreplane. In some embodiments, the light detection modules may be orientedwith respect to each other (as referenced in an X-Z plane) at an angleranging from 0° to 180°, such as from 10° to 170°, such as from 20° to160°, such as from 25° to 150°, such as from 30° to 120° and includingfrom 45° to 90°. In embodiments, the light detection modules may bearranged with respect to each other at an angle that is the same ordifferent depending on the number of light detection modules in thelight detection system. For example, in certain instances the anglebetween a first light detection module and a second light detectionmodule is the same as the angle between the second light detectionmodules and a third light detection module. In some embodiments, theangle between a first light detection module and a second lightdetection module are different than the angle between the second lightdetection module and a third light detection module. In someembodiments, the light detection modules are positioned in a geometricarrangement such as a star-shaped configuration, a triangularconfiguration, a square configuration, rectangular configuration,trapezoidal configuration, triangular configuration, hexagonalconfiguration, heptagonal configuration, octagonal configuration,nonagonal configuration, decagonal configuration, dodecagonalconfiguration, circular configuration, oval configuration as well asirregular shaped configurations.

In some embodiments, each light detection module includes an opticaladjustment component configured to convey light having a predeterminedsub-spectral range to one or more photodetectors. By “opticaladjustment” is meant that light is changed or adjusted when conveyed toeach photodetector in the light detection module. In some embodiments,optical adjustment includes propagating light having a predeterminedsub-spectral range to a photodetector. In some embodiments, each lightdetection module includes one or more optical adjustment components thatare configured to separate light conveyed from the wavelength separatorinto predetermined sub-spectral ranges by passing light having apredetermined sub-spectral range and reflecting one or more remainingspectral ranges of light. In other embodiments, the optical adjustmentcomponent is configured to separate light conveyed from the wavelengthseparator into predetermined sub-spectral ranges by passing light havinga predetermined sub-spectral range and absorbing one or more remainingspectral ranges of light. In yet other embodiments, the opticaladjustment component is configured to spatially diffract light conveyedfrom the wavelength separator into the predetermined sub-spectralranges. Optical adjustment components may be any convenient lightseparation protocol, such as one or more dichroic mirrors, bandpassfilters, diffraction gratings, beam splitters or prisms. In certainembodiments, optical adjustment components in the light detectionmodules that are configured to separate light conveyed from thewavelength separator into predetermined sub-spectral ranges are dichroicmirrors.

Depending on the wavelengths of light passed from the wavelengthseparator to the light detection module (as described above), the one ormore optical components in the light detection module may be configuredto convey light having wavelengths that range from a first wavelength,Y_(i) (in nanometers, nm) to a second wavelength Y_(n) (in nanometers,nm) to the photodetectors. In some embodiments, the one or more opticalcomponents are configured to convey light having wavelengths that rangefrom 100 nm to 1500 nm to the photodetectors, such as from 150 nm to1450 nm, such as from 200 nm to 1400 nm, such as from 250 nm to 1350 nm,such as from 300 nm to 1300 nm, such as from 350 nm to 1250 nm, such asfrom 400 nm to 1200 nm, such as from 450 nm to 1150 nm, such as from 500nm to 1100 nm, such as from 550 nm to 1050 nm and including propagatinglight having wavelengths that range from 600 nm to 1000 nm to thephotodetectors.

In embodiments, the optical components in each light detection moduleare configured to convey a predetermined sub-spectral range of light,Y_(s) (in nanometers, nm) to each photodetector. The predeterminedsub-spectral ranges conveyed by each optical component may vary, wherecertain optical components of interest are configured to conveysub-spectral ranges of light that span from 5 nm to 50 nm, such as from6 nm to 49 nm, such as from 7 nm to 48 nm, such as from 8 nm to 47 nm,such as from 9 nm to 46 nm and including from nm to 45 nm. In certainembodiments, the optical component is configured to pass a spectralrange of light that spans 20 nm.

For instance, in one example the one or more optical components areconfigured to pass light having wavelengths that range from 360 nm(i.e., Y_(i)=360 nm) to a 480 nm (i.e., Y_(n)=480 nm) in sub-spectralranges that span 20 nm (i.e., Y_(s)=20 nm). In this embodiment, thelight detection module includes a first optical component that isconfigured to convey light having wavelengths that range from 360 nm to380 nm to a photodetector; a second optical component that is configuredto convey light having wavelengths that range from 380 nm to 400 nm to aphotodetector; a third optical component that is configured to conveylight having wavelengths that range from 400 nm to 420 nm to aphotodetector; a fourth optical component that is configured to conveylight having wavelengths that range from 420 nm to 440 nm to aphotodetector; a fifth optical component that is configured to conveylight having wavelengths that range from 440 nm to 460 nm to aphotodetector; and a sixth optical component that is configured toconvey light having wavelengths that range from 460 nm to 480 nm to aphotodetector.

In some embodiments, the optical components in each light detectionmodule are in optical communication with each other, such as beingpositioned to convey light between each other. The optical componentsmay be oriented with respect to each other in the light detection module(as referenced in an X-Z plane) at an angle ranging from 10° to 180°,such as from 15° to 170°, such as from 20° to 160°, such as from 25° to150°, such as from 30° to 120° and including from 45° to 90°. In someinstances, the optical components are positioned along a single plane.In other instances, the optical components are positioned along morethan one plane. For example, the optical components may be positionedalong two or more parallel planes, such as three or more, such as fouror more and including five or more parallel planes. In certaininstances, the optical components are arranged into a geometricconfiguration, where arrangements of interest include, but are notlimited to a square configuration, rectangular configuration,trapezoidal configuration, triangular configuration, hexagonalconfiguration, heptagonal configuration, octagonal configuration,nonagonal configuration, decagonal configuration, dodecagonalconfiguration, circular configuration, oval configuration as well asirregular shaped configurations. In certain embodiments, the opticalcomponents are arranged in a hexagonal configuration. In otherembodiments, the optical components are arranged in a heptagonalconfiguration.

In some embodiments, the optical components are configured to conveylight between each other. In some instances, each optical component isconfigured to pass a spectral range of light and to convey (e.g., byreflection) one or more remaining spectral ranges of light to anotheroptical component. In one example, the light detection module includes 3optical components. The first optical component is configured to receivelight from a wavelength separator and to pass a first sub-spectral rangeof light and convey a second sub-spectral range of light to the secondoptical component. The second optical component is configured to pass athird sub-spectral range of light and to convey a fourth sub-spectralrange of light to the third optical component. In some instances, thethird sub-spectral range of light is a portion of the secondsub-spectral range of light, such as a sub-spectral range that spans 90%or less of the second sub-spectral range of light, such 85% or less,such as 80% or less, such as 75% or less, such as 70% or less, such as65% or less, such as 60% or less, such as 55% or less, such as 50%. Thethird optical component is configured to pass a fifth sub-spectral rangeof light. In some instances, the fifth sub-spectral range of light is aportion of the fourth sub-spectral range of light, such as asub-spectral range that spans 90% or less of the fourth sub-spectralrange of light, such 85% or less, such as 80% or less, such as 75% orless, such as 70% or less, such as 65% or less, such as 60% or less,such as 55% or less, such as 50%.

In another example, the light detection module includes 5 opticalcomponents. The first optical component is configured to receive lightfrom a wavelength separator and to pass a first sub-spectral range oflight and convey a second sub-spectral range of light to the secondoptical component. The second optical component is configured to pass athird sub-spectral range of light and to convey a fourth sub-spectralrange of light to the third optical component. In some instances, thethird sub-spectral range of light is a portion of the secondsub-spectral range of light, such as a sub-spectral range that spans 90%or less of the second sub-spectral range of light, such 85% or less,such as 80% or less, such as 75% or less, such as 70% or less, such as65% or less, such as 60% or less, such as 55% or less, such as 50%. Thethird optical component is configured to pass a fifth sub-spectral rangeof light and to convey a sixth sub-spectral range of light to the fourthoptical component. In some instances, the fifth sub-spectral range oflight is a portion of the fourth sub-spectral range of light, such as asub-spectral range that spans 90% or less of the fourth sub-spectralrange of light, such 85% or less, such as 80% or less, such as 75% orless, such as 70% or less, such as 65% or less, such as 60% or less,such as 55% or less, such as 50%. The fourth optical component isconfigured to pass a seventh sub-spectral range of light and to conveyan eighth sub-spectral range of light to the fifth optical component. Insome instances, the seventh spectral range of light is a portion of thesixth spectral range of light, such as a spectral range that spans 90%or less of the sixth spectral range of light, such 85% or less, such as80% or less, such as 75% or less, such as 70% or less, such as 65% orless, such as 60% or less, such as 55% or less, such as 50%. The fifthoptical component is configured to pass a ninth sub-spectral range oflight. In some instances, the ninth sub-spectral range of light is aportion of the eighth sub-spectral range of light, such as asub-spectral range that spans 90% or less of the eighth sub-spectralrange of light, such 85% or less, such as 80% or less, such as 75% orless, such as 70% or less, such as 65% or less, such as 60% or less,such as 55% or less, such as 50%.

FIG. 3 depicts components of a light detection module positioned alongtwo parallel planes according to certain embodiments. A spectral rangeof light SRx from a wavelength separator (as described above) having aset of wavelengths 301 (e.g., 360 nm to 480 nm) is conveyed to a firstoptical component OA1 configured to pass a first sub-spectral range oflight sSR1 (e.g., 360 nm to 380 nm) and convey light having a set ofwavelengths 302 (e.g., 380 nm to 480 nm) to a second optical componentOA2. The first sub-spectral range of light sSR1 is conveyed to a firstphotodetector D1. The second optical component OA2 is configured to passa second sub-spectral range of light sSR2 (e.g., 380 nm to 400 nm) andconvey light having a set of wavelengths 303 (e.g., 400 nm to 480 nm) toa third optical component OA3. The second sub-spectral range of lightsSR2 is conveyed to a second photodetector D2. The third opticalcomponent OA3 is configured to pass a third sub-spectral range of lightsSR3 (e.g., 400 nm to 420 nm) and convey light having a set ofwavelengths 304 (e.g., 420 nm to 480 nm) to a fourth optical componentOA4. The third sub-spectral range of light sSR3 is conveyed to a thirdphotodetector D3. The fourth optical component OA4 is configured to passa fourth sub-spectral range of light sSR4 (e.g., 420 nm to 440 nm) andconvey light having a set of wavelengths 305 (e.g., 440 nm to 480 nm) toa fifth optical component OA5. The fourth sub-spectral range of lightsSR4 is conveyed to a fourth photodetector D4. The fifth opticalcomponent OA5 is configured to pass a fifth sub-spectral range of lightsSR5 (e.g., 440 nm to 460 nm) and convey light having a set ofwavelengths 306 (e.g., 460 nm to 480 nm) to a sixth optical componentOA6. The fifth sub-spectral range of light sSR5 is conveyed to a fifthphotodetector D5. The sixth optical component OA6 is configured to passa sixth sub-spectral range of light sSR6 (e.g., 460 nm to 480 nm). Thesixth sub-spectral range of light sSR6 is conveyed to a sixthphotodetector D6. In this embodiment, light is conveyed along asequential zig-zag light path.

FIG. 4 depicts components of a light detection module positioned in apolygonal configuration according to certain embodiments. A spectralrange of light SRx from a wavelength separator (as described above)having a set of wavelengths 401 (e.g., 200 nm to 500 nm) is conveyed toa first optical component OA1 configured to pass a first sub-spectralrange of light sSR1 (e.g., 200 nm to 250 nm) and convey light having aset of wavelengths 402 (e.g., 250 nm to 500 nm) to a second opticalcomponent OA2. The first sub-spectral range of light sSR1 is conveyed toa first photodetector D1. The second optical component OA2 is configuredto pass a second sub-spectral range of light sSR2 (e.g., 250 nm to 300nm) and convey light having a set of wavelengths 403 (e.g., 300 nm to500 nm) to a third optical component OA3. The second sub-spectral rangeof light sSR2 is conveyed to a second photodetector D2. The thirdoptical component OA3 is configured to pass a third sub-spectral rangeof light sSR3 (e.g., 300 nm to 350 nm) and convey light having a set ofwavelengths 404 (e.g., 350 nm to 500 nm) to a fourth optical componentOA4. The third sub-spectral range of light sSR3 is conveyed to a thirdphotodetector D3. The fourth optical component OA4 is configured to passa fourth sub-spectral range of light sSR4 (e.g., 350 nm to 400 nm) andconvey light having a set of wavelengths 405 (e.g., 400 nm to 500 nm) toa fifth optical component OA5. The fourth sub-spectral range of lightsSR4 is conveyed to a fourth photodetector D4. The fifth opticalcomponent OA5 is configured to pass a fifth sub-spectral range of lightsSR5 (e.g., 400 nm to 450 nm) and convey light having a set ofwavelengths 406 (e.g., 450 nm to 500 nm) to a sixth optical componentOA6. The fifth sub-spectral range of light sSR5 is conveyed to a fifthphotodetector D5. The sixth optical component OA6 is configured to passa sixth sub-spectral range of light sSR6 (e.g., 450 nm to 500 nm). Thesixth sub-spectral range of light sSR6 is conveyed to a sixthphotodetector D6. In this embodiment, light is conveyed along astar-shaped light path.

FIG. 5 depicts components of a light detection module positioned alongtwo parallel planes according to certain embodiments. A spectral rangeof light SRx from a wavelength separator (as described above) having aset of wavelengths 501 (e.g., 360 nm to 600 nm) is conveyed to a firstoptical component OA1 configured to pass a first sub-spectral range oflight sSR1 (e.g., 360 nm to 400 nm) and convey light having a set ofwavelengths 502 (e.g., 400 nm to 600 nm) to a second optical componentOA2. The first sub-spectral range of light sSR1 is conveyed to a firstphotodetector D1. The second optical component OA2 is configured to passa second sub-spectral range of light sSR2 (e.g., 400 nm to 440 nm) andconvey light having a set of wavelengths 503 (e.g., 440 nm to 600 nm) toa third optical component OA3. The second sub-spectral range of lightsSR2 is conveyed to a second photodetector D2. The third opticalcomponent OA3 is configured to pass a third sub-spectral range of lightsSR3 (e.g., 440 nm to 480 nm) and convey light having a set ofwavelengths 504 (e.g., 480 nm to 600 nm) to a fourth optical componentOA4. The third sub-spectral range of light sSR3 is conveyed to a thirdphotodetector D3. The fourth optical component OA4 is configured to passa fourth sub-spectral range of light sSR4 (e.g., 480 nm to 520 nm) andconvey light having a set of wavelengths 505 (e.g., 520 nm to 600 nm) toa fifth optical component OA5. The fourth sub-spectral range of lightsSR4 is conveyed to a fourth photodetector D4. The fifth opticalcomponent OA5 is configured to pass a fifth sub-spectral range of lightsSR5 (e.g., 520 nm to 560 nm) and convey light having a set ofwavelengths 506 (e.g., 560 nm to 600 nm) to a sixth optical componentOA6. The fifth sub-spectral range of light sSR5 is conveyed to a fifthphotodetector D5. The sixth optical component OA6 is configured to passa sixth sub-spectral range of light sSR6 (e.g., 560 nm to 600 nm). Thesixth sub-spectral range of light sSR6 is conveyed to a sixthphotodetector D6. In this embodiment, light is conveyed along aback-and-forth zig-zag light path.

As described above, light detection systems are configured to generate aplurality of sub-spectral ranges of light from the light collected fromthe sample. In some embodiments, light detection systems are configuredto generate 2 or more distinct spectral ranges of light for everyreflection by an optical component (e.g., dichroic mirror) in the lightdetection system, such as 3 or more, such as 4 or more and includingbeing configured to generate 5 or more distinct spectral ranges of lightfor every reflection by an optical component in the subject lightdetection systems. In certain embodiments, light detection systems areconfigured to generate 30 distinct spectral ranges using 10 reflectionsby optical components or less, such as generating 30 distinct spectralranges of light from 9 reflections by optical components or less. Incertain instances, the ratio of generated distinct spectral ranges tonumber of reflections by optical components in the subject lightdetection systems may range from 2:1 to 10:1, such as from 3:1 to 7:1and including from 3:1 to 5:1. In some instances, the optical componentis configured to collimate the light. The term “collimate” is used inits conventional sense to refer to the optically adjusting thecollinearity of light propagation or reducing divergence by the light offrom a common axis of propagation. In some instances, collimatingincludes narrowing the spatial cross section of a light beam. In otherinstances, optical includes changing the direction of the light beam,such as changing the propagation of the light beam by 1° or more, suchas by 5° or more, such as by 10° or more, such as by 15° or more, suchas by 20° or more, such as by 25° or more, such as by 30° or more, suchas by 45° or more, such as by 60° or more, such as by 75° or more andincluding changing the direction of light propagation by 90° or more. Inyet other instances, optical is a de-magnification protocol so as todecrease the dimensions of the light (e.g., beam spot), such asdecreasing the dimensions by 5% or more, such as by 10% or more, such asby 25% or more, such as by 50% or more and including decreasing thedimensions by 75% or more.

Each sub-spectral range of light is conveyed by the optical component toa photodetector. In some embodiments, the optical component is inphysical contact with the photodetector. In other embodiments, theoptical component is in optical communication with the active surface ofthe photodetector and may be positioned 0.001 mm or more from thephotodetector, such as 0.005 mm or more, such as 0.01 mm or more, suchas 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, suchas 1 mm or more, such as 10 mm or more, such as 25 mm or more, such as50 mm or more and including 100 mm or more from the photodetector.

Photodetectors may be releasably connected to each optical component inthe subject light detection modules. Photodetectors and opticalcomponents may be connected by any convenient protocol. In certainembodiments, the photodetectors and optical components are connectedtogether by co-mounting the photodetector with the optical component orwith a fastener, such as a hook and loop fasteners, magnets, latches,notches, countersinks, counter-bores, grooves, pins, tethers, hinges,Velcro, non-permanent adhesives or a combination thereof. In certaininstances, a photodetector is connected to an optical component byslot-fitting the wavelength separator into a groove of the lightdetection module. In yet other instances, a photodetector is connectedto an optical component by one or more screws.

In embodiments, each light detection module includes two or morephotodetectors, such as 3 or more, such as 4 or more, such as 5 or more,such as 6 or more, such as 7 or more, such as 8 or more, such as 9 ormore, such as or more, such as 15 or more, such as 25 or more, such as50 or more and including 100 or more photodetectors. In someembodiments, light detection modules include one or more photodetectorarrays. The term “photodetector array” is used in its conventional senseto refer to an arrangement or series of two or more photodetectors. Inembodiments, photodetector arrays may include 2 or more photodetectors,such as 3 or more photodetectors, such as 4 or more photodetectors, suchas 5 or more photodetectors, such as 6 or more photodetectors, such as 7or more photodetectors, such as 8 or more photodetectors, such as 9 ormore photodetectors, such as 10 or more photodetectors, such as 12 ormore photodetectors and including 15 or more photodetectors. Thephotodetectors in each array may be arranged in any geometricconfiguration as desired, where arrangements of interest include, butare not limited to a square configuration, rectangular configuration,trapezoidal configuration, triangular configuration, hexagonalconfiguration, heptagonal configuration, octagonal configuration,nonagonal configuration, decagonal configuration, dodecagonalconfiguration, circular configuration, oval configuration as well asirregular shaped configurations. The photodetectors in eachphotodetector array may be oriented with respect to the other (asreferenced in an X-Z plane) at an angle ranging from 10° to 180°, suchas from to 170°, such as from 20° to 160°, such as from 25° to 150°,such as from to 120° and including from 45° to 90°.

The photodetectors may be any convenient optical sensor, such asactive-pixel sensors (APSs), avalanche photodiode, image sensors,charge-coupled devices (CCDs), intensified charge-coupled devices(ICCDs), complementary metal-oxide semiconductor (CMOS) image sensors orN-type metal-oxide semiconductor (NMOS) image sensors, light emittingdiodes, photon counters, bolometers, pyroelectric detectors,photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes,phototransistors, quantum dot photoconductors or photodiodes andcombinations thereof, among other types of photodetectors. In certainembodiments, photodetectors include photomultiplier tubes, such as metalpackage photomultiplier tubes.

Photodetectors of interest are configured to measure collected light atone or more wavelengths, such as at 2 or more wavelengths, such as at 5or more different wavelengths, such as at 10 or more differentwavelengths, such as at 25 or more different wavelengths, such as at 50or more different wavelengths, such as at 100 or more differentwavelengths, such as at 200 or more different wavelengths, such as at300 or more different wavelengths and including measuring light emittedby a sample in the flow stream at 400 or more different wavelengths.

In embodiments, the photodetectors are configured to measure lightcontinuously or in discrete intervals. In some instances, photodetectorsof interest are configured to take measurements of the collected lightcontinuously. In other instances, the photodetectors are configured totake measurements in discrete intervals, such as measuring light every0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every1 millisecond, every 10 milliseconds, every 100 milliseconds andincluding every 1000 milliseconds, or some other interval.

FIG. 6 depicts a light detection system having a plurality of wavelengthseparators and light detection modules according to certain embodiments.Light from a sample Light from a sample is conveyed through an opticalcollection system OC having an optical component configured to passlight having a set of wavelengths 601 to a first wavelength separatorWS1 that is configured to pass a first spectral range of light SR1 andconvey light having a set of wavelengths 602 to a second wavelengthseparator WS2. The light of spectral range SR1 is conveyed to a firstlight detection module LDM1. The second wavelength separator WS2 isconfigured to pass a second spectral range of light SR2 and convey lighthaving a set of wavelengths 603 to a third wavelength separator WS3. Thelight of spectral range SR2 is conveyed to a second light detectionmodule LDM2. The third wavelength separator WS3 is configured to pass athird spectral range of light SR3 and convey light having a set ofwavelengths 604 to a fourth wavelength separator WS4. The light ofspectral range SR3 is conveyed to a third light detection module LDM3.The fourth wavelength separator WS4 is configured to pass a fourthspectral range of light SR4 and convey light having a fifth spectralrange of light SR5 to a fifth light detection module LDM5. The light ofspectral range SR4 is conveyed to a fourth light detection module LDM4.In this embodiment, each of spectral ranges of light SR1, SR2, SR3, SR4and SR5 are conveyed to light detection modules, LDM1, LDM2, LDM3, LDM4and LDM5, respectively, which have a back-and-forth zig-zagconfiguration as described in FIG. 5 above.

FIG. 7 depicts light detection systems having 3 or more concentricallyarranged wavelength separators optically coupled to light detectionmodules according to certain embodiments. FIG. 7A depicts a lightdetection system having three wavelength separators 711 a, 711 b and 711c. Each wavelength separator is optically coupled to a light detectionmodule, 712 a, 712 b and 712 c. FIG. 7B depicts a light detection systemhaving four wavelength separators 721 a, 721 b, 721 c and 721 d. Eachwavelength separator is optically coupled to a light detection module,722 a, 722 b, 722 c and 722 d. FIG. 7C depicts a light detection systemhaving five wavelength separators 731 a, 731 b, 731 c, 731 d and 731 e.Each wavelength separator is optically coupled to a light detectionmodule, 732 a, 732 b, 732 c, 732 d and 732 e. FIG. 7D depicts a lightdetection system having six wavelength separators 741 a, 741 b, 741 c,741 d, 741 e and 741 f. Each wavelength separator is optically coupledto a light detection module, 742 a, 742 b, 742 c, 742 d, 742 e and 742f. FIG. 7E depicts a three dimensional view of the light detectionsystem of FIG. 7D.

Systems for Measuring Light Emitted by a Sample

Aspects of the present disclosure also include systems for measuringlight from a sample (e.g., in the flow stream in a flow cytometer). Incertain embodiments, systems include a light source and a clusteredwavelength division light detection system having three or morewavelength separators that are each configured to pass light having apredetermined spectral range and one or more light detection modules inoptical communication with each wavelength separator having a pluralityof photodetectors and an optical component that conveys light having apredetermined sub-spectral range to the photodetectors, as describedabove. In some embodiments, the system is a flow cytometer. In someinstances, the light detection system is non-releasably integrated intothe flow cytometer. In certain embodiments, the light detection systemis in optical communication with the source of sample (e.g., the flowstream in a flow cytometer) through an optical collection system (e.g.,fiber optics or free-space light relay system).

Systems of interest for measuring light from a sample include a lightsource. In embodiments, the light source may be any suitable broadbandor narrow band source of light. Depending on the components in thesample (e.g., cells, beads, non-cellular particles, etc.), the lightsource may be configured to emit wavelengths of light that vary, rangingfrom 200 nm to 1500 nm, such as from 250 nm to 1250 nm, such as from 300nm to 1000 nm, such as from 350 nm to 900 nm and including from 400 nmto 800 nm. For example, the light source may include a broadband lightsource emitting light having wavelengths from 200 nm to 900 nm. In otherinstances, the light source includes a narrow band light source emittinga wavelength ranging from 200 nm to 900 nm. For example, the lightsource may be a narrow band LED (1 nm-25 nm) emitting light having awavelength ranging between 200 nm to 900 nm.

In some embodiments, the light source is a laser. Lasers of interest mayinclude pulsed lasers or continuous wave lasers. For example, the lasermay be a gas laser, such as a helium-neon laser, argon laser, kryptonlaser, xenon laser, nitrogen laser, CO₂ laser, CO laser, argon-fluorine(ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenonchlorine (XeCl) excimer laser or xenon-fluorine (XeF) excimer laser or acombination thereof; a dye laser, such as a stilbene, coumarin orrhodamine laser; a metal-vapor laser, such as a helium-cadmium (HeCd)laser, helium-mercury (HeHg) laser, helium-selenium (HeSe) laser,helium-silver (HeAg) laser, strontium laser, neon-copper (NeCu) laser,copper laser or gold laser and combinations thereof; a solid-statelaser, such as a ruby laser, an Nd:YAG laser, NdCrYAG laser, Er:YAGlaser, Nd:YLF laser, Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser,titanium sapphire laser, thulim YAG laser, ytterbium YAG laser,ytterbium₂O₃ laser or cerium doped lasers and combinations thereof; asemiconductor diode laser, optically pumped semiconductor laser (OPSL),or a frequency doubled- or frequency tripled implementation of any ofthe above mentioned lasers.

In other embodiments, the light source is a non-laser light source, suchas a lamp, including but not limited to a halogen lamp, deuterium arclamp, xenon arc lamp, a light-emitting diode, such as a broadband LEDwith continuous spectrum, superluminescent emitting diode, semiconductorlight emitting diode, wide spectrum LED white light source, an multi-LEDintegrated. In some instances the non-laser light source is a stabilizedfiber-coupled broadband light source, white light source, among otherlight sources or any combination thereof.

In certain embodiments, the light source is a light beam generator thatis configured to generate two or more beams of frequency shifted light.In some instances, the light beam generator includes a laser, aradiofrequency generator configured to apply radiofrequency drivesignals to an acousto-optic device to generate two or more angularlydeflected laser beams. In these embodiments, the laser may be a pulsedlasers or continuous wave laser. For example lasers in light beamgenerators of interest may be a gas laser, such as a helium-neon laser,argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, COlaser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF)excimer laser, xenon chlorine (XeCl) excimer laser or xenon-fluorine(XeF) excimer laser or a combination thereof; a dye laser, such as astilbene, coumarin or rhodamine laser; a metal-vapor laser, such as ahelium-cadmium (HeCd) laser, helium-mercury (HeHg) laser,helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontiumlaser, neon-copper (NeCu) laser, copper laser or gold laser andcombinations thereof; a solid-state laser, such as a ruby laser, anNd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser, Nd:YVO4 laser,Nd:YCa₄O(BO3)3 laser, Nd:YCOB laser, titanium sapphire laser, thulim YAGlaser, ytterbium YAG laser, ytterbium2O3 laser or cerium doped lasersand combinations thereof.

The acousto-optic device may be any convenient acousto-optic protocolconfigured to frequency shift laser light using applied acoustic waves.In certain embodiments, the acousto-optic device is an acousto-opticdeflector. The acousto-optic device in the subject system is configuredto generate angularly deflected laser beams from the light from thelaser and the applied radiofrequency drive signals. The radiofrequencydrive signals may be applied to the acousto-optic device with anysuitable radiofrequency drive signal source, such as a direct digitalsynthesizer (DDS), arbitrary waveform generator (AWG), or electricalpulse generator.

In embodiments, a controller is configured to apply radiofrequency drivesignals to the acousto-optic device to produce the desired number ofangularly deflected laser beams in the output laser beam, such as beingconfigured to apply 3 or more radiofrequency drive signals, such as 4 ormore radiofrequency drive signals, such as 5 or more radiofrequencydrive signals, such as 6 or more radiofrequency drive signals, such as 7or more radiofrequency drive signals, such as 8 or more radiofrequencydrive signals, such as 9 or more radiofrequency drive signals, such as10 or more radiofrequency drive signals, such as 15 or moreradiofrequency drive signals, such as 25 or more radiofrequency drivesignals, such as 50 or more radiofrequency drive signals and includingbeing configured to apply 100 or more radiofrequency drive signals.

In some instances, to produce an intensity profile of the angularlydeflected laser beams in the output laser beam, the controller isconfigured to apply radiofrequency drive signals having an amplitudethat varies such as from about 0.001 V to about 500 V, such as fromabout 0.005 V to about 400 V, such as from about 0.01 V to about 300 V,such as from about 0.05 V to about 200 V, such as from about 0.1 V toabout 100 V, such as from about 0.5 V to about 75 V, such as from about1 V to 50 V, such as from about 2 V to 40 V, such as from 3 V to about30 V and including from about 5 V to about 25 V. Each appliedradiofrequency drive signal has, in some embodiments, a frequency offrom about 0.001 MHz to about 500 MHz, such as from about 0.005 MHz toabout 400 MHz, such as from about 0.01 MHz to about 300 MHz, such asfrom about 0.05 MHz to about 200 MHz, such as from about 0.1 MHz toabout 100 MHz, such as from about 0.5 MHz to about 90 MHz, such as fromabout 1 MHz to about 75 MHz, such as from about 2 MHz to about 70 MHz,such as from about 3 MHz to about 65 MHz, such as from about 4 MHz toabout 60 MHz and including from about 5 MHz to about 50 MHz.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam with angularly deflectedlaser beams having a desired intensity profile. For example, the memorymay include instructions to produce two or more angularly deflectedlaser beams with the same intensities, such as 3 or more, such as 4 ormore, such as 5 or more, such as 10 or more, such as 25 or more, such as50 or more and including memory may include instructions to produce 100or more angularly deflected laser beams with the same intensities. Inother embodiments, the may include instructions to produce two or moreangularly deflected laser beams with different intensities, such as 3 ormore, such as 4 or more, such as 5 or more, such as 10 or more, such as25 or more, such as 50 or more and including memory may includeinstructions to produce 100 or more angularly deflected laser beams withdifferent intensities.

In certain embodiments, the controller has a processor having memoryoperably coupled to the processor such that the memory includesinstructions stored thereon, which when executed by the processor, causethe processor to produce an output laser beam having increasingintensity from the edges to the center of the output laser beam alongthe horizontal axis. In these instances, the intensity of the angularlydeflected laser beam at the center of the output beam may range from0.1% to about 99% of the intensity of the angularly deflected laserbeams at the edge of the output laser beam along the horizontal axis,such as from 0.5% to about 95%, such as from 1% to about 90%, such asfrom about 2% to about 85%, such as from about 3% to about 80%, such asfrom about 4% to about 75%, such as from about 5% to about 70%, such asfrom about 6% to about 65%, such as from about 7% to about 60%, such asfrom about 8% to about 55% and including from about 10% to about 50% ofthe intensity of the angularly deflected laser beams at the edge of theoutput laser beam along the horizontal axis. In other embodiments, thecontroller has a processor having memory operably coupled to theprocessor such that the memory includes instructions stored thereon,which when executed by the processor, cause the processor to produce anoutput laser beam having an increasing intensity from the edges to thecenter of the output laser beam along the horizontal axis. In theseinstances, the intensity of the angularly deflected laser beam at theedges of the output beam may range from 0.1% to about 99% of theintensity of the angularly deflected laser beams at the center of theoutput laser beam along the horizontal axis, such as from 0.5% to about95%, such as from 1% to about 90%, such as from about 2% to about 85%,such as from about 3% to about 80%, such as from about 4% to about 75%,such as from about 5% to about 70%, such as from about 6% to about 65%,such as from about 7% to about 60%, such as from about 8% to about 55%and including from about 10% to about 50% of the intensity of theangularly deflected laser beams at the center of the output laser beamalong the horizontal axis. In yet other embodiments, the controller hasa processor having memory operably coupled to the processor such thatthe memory includes instructions stored thereon, which when executed bythe processor, cause the processor to produce an output laser beamhaving an intensity profile with a Gaussian distribution along thehorizontal axis. In still other embodiments, the controller has aprocessor having memory operably coupled to the processor such that thememory includes instructions stored thereon, which when executed by theprocessor, cause the processor to produce an output laser beam having atop hat intensity profile along the horizontal axis.

In embodiments, light beam generators of interest may be configured toproduce angularly deflected laser beams in the output laser beam thatare spatially separated. Depending on the applied radiofrequency drivesignals and desired irradiation profile of the output laser beam, theangularly deflected laser beams may be separated by 0.001 μm or more,such as by 0.005 μm or more, such as by 0.01 μm or more, such as by 0.05μm or more, such as by 0.1 μm or more, such as by 0.5 μm or more, suchas by 1 μm or more, such as by 5 μm or more, such as by 10 μm or more,such as by 100 μm or more, such as by 500 μm or more, such as by 1000 μmor more and including by 5000 μm or more. In some embodiments, systemsare configured to produce angularly deflected laser beams in the outputlaser beam that overlap, such as with an adjacent angularly deflectedlaser beam along a horizontal axis of the output laser beam. The overlapbetween adjacent angularly deflected laser beams (such as overlap ofbeam spots) may be an overlap of 0.001 μm or more, such as an overlap of0.005 μm or more, such as an overlap of 0.01 μm or more, such as anoverlap of 0.05 μm or more, such as an overlap of 0.1 μm or more, suchas an overlap of 0.5 μm or more, such as an overlap of 1 μm or more,such as an overlap of 5 μm or more, such as an overlap of 10 μm or moreand including an overlap of 100 μm or more.

In certain instances, light beam generators configured to generate twoor more beams of frequency shifted light include laser excitationmodules as described in U.S. Pat. Nos. 9,423,353; 9,784,661 and10,006,852 and U.S. Patent Publication Nos. 2017/0133857 and2017/0350803, the disclosures of which are herein incorporated byreference.

The light source may be positioned any suitable distance from the sample(e.g., the flow stream in a flow cytometer), such as at a distance of0.001 mm or more from the flow stream, such as 0.005 mm or more, such as0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, suchas 0.5 mm or more, such as 1 mm or more, such as 5 mm or more, such as10 mm or more, such as 25 mm or more and including at a distance of 100mm or. In addition, the light source irradiate the sample at anysuitable angle (e.g., relative the vertical axis of the flow stream),such as at an angle ranging from 10° to 90°, such as from 15° to 85°,such as from 20° to 80°, such as from 25° to 75° and including from 30°to 60°, for example at a 90° angle.

The light source may be configured to irradiate the sample continuouslyor in discrete intervals. In some instances, systems include a lightsource that is configured to irradiate the sample continuously, such aswith a continuous wave laser that continuously irradiates the flowstream at the interrogation point in a flow cytometer. In otherinstances, systems of interest include a light source that is configuredto irradiate the sample at discrete intervals, such as every 0.001milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1millisecond, every 10 milliseconds, every 100 milliseconds and includingevery 1000 milliseconds, or some other interval. Where the light sourceis configured to irradiate the sample at discrete intervals, systems mayinclude one or more additional components to provide for intermittentirradiation of the sample with the light source. For example, thesubject systems in these embodiments may include one or more laser beamchoppers, manually or computer controlled beam stops for blocking andexposing the sample to the light source.

In embodiments, light emitted by the sample is conveyed to the subjectlight detection systems (as described above), having two or morephotodetector arrays. As described above, photodetectors in the subjectphotodetectors may include, but are not limited to optical sensors, suchas active-pixel sensors (APSs), avalanche photodiode, image sensors,charge-coupled devices (CCDs), intensified charge-coupled devices(ICCDs), light emitting diodes, photon counters, bolometers,pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes,photomultiplier tubes, phototransistors, quantum dot photoconductors orphotodiodes and combinations thereof, among other photodetectors. Forexample, the light collection system for measuring light from a samplemay include photodetectors arrays having 2 photodetectors or more, suchas 3 photodetectors or more, such as 4 photodetectors or more, such as 5photodetectors or more, such as 10 photodetectors or more, such as 25photodetectors or more and including 50 photodetectors or more. Incertain embodiments, systems include photodetector arrays with 5photodetectors.

In embodiments of the present disclosure, detectors of interest areconfigured to measure collected light at one or more wavelengths, suchas at 2 or more wavelengths, such as at 5 or more different wavelengths,such as at 10 or more different wavelengths, such as at 25 or moredifferent wavelengths, such as at 50 or more different wavelengths, suchas at 100 or more different wavelengths, such as at 200 or moredifferent wavelengths, such as at 300 or more different wavelengths andincluding measuring light emitted by a sample in the flow stream at 400or more different wavelengths.

In embodiments, the photodetectors of the light detection system areconfigured to measure light continuously or in discrete intervals. Insome instances, detectors of interest are configured to takemeasurements of the collected light continuously. In other instances,detectors of interest are configured to take measurements in discreteintervals, such as measuring light every 0.001 millisecond, every 0.01millisecond, every 0.1 millisecond, every 1 millisecond, every 10milliseconds, every 100 milliseconds and including every 1000milliseconds, or some other interval.

In some embodiments, systems for measuring light from sample include aoptical collection system for collecting and directing light from thesample source (e.g., flow stream) to the subject light detectionssystems. The optical collection system may be physically coupled to thelight detection system, such as with an adhesive, co-molded together orintegrated into the light detection system. In certain embodiments, theoptical collection system and the light detection system are integratedinto a single unit. In other embodiments, the optical collection systemis coupled to the light detection system with an connector, such as witha hook and loop fasteners, magnets, latches, notches, countersinks,counter-bores, grooves, pins, tethers, hinges, Velcro, non-permanentadhesives or a combination thereof.

In other embodiments, the light detection system and the opticalcollection system are in optical communication, but are not physicallyin contact. For example, the optical collection system may be positioned0.001 mm or more from the light detection system, such as 0.005 mm ormore, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mmor more, such as 0.5 mm or more, such as 1 mm or more, such as 10 mm ormore, such as 25 mm or more, such as 50 mm or more and including 100 mmor more from the light detection system.

In some embodiments, the optical collection system includes fiberoptics. For example, in some instances the optical collection system maybe a fiber optics light relay bundle and light is conveyed through thefiber optics light relay bundle to the light detection system. In otherembodiments, the optical collection system is a free-space light relaysystem. For instance, the free-space light relay system may include ahousing having a proximal end and a distal end, the proximal end beingcoupled to the light detection system. The free-space relay system mayinclude any combination of different optical components, such as one ormore lenses, mirrors, slits, pinholes, wavelength separators, or acombination thereof.

In certain embodiments, the subject systems are flow cytometric systemsemploying the above described light detection system for detecting lightemitted by a sample in a flow stream. In certain embodiments, thesubject systems are flow cytometric systems. Suitable flow cytometrysystems may include, but are not limited to those described in Ormerod(ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997);Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in MolecularBiology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed.,Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. January; 49(pt1):17-28; Linden, et. al., Semin Throm Hemost. 2004 October;30(5):502-11; Alison, et al. J Pathol, 2010 December; 222(4):335-344;and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst.24(3):203-255; the disclosures of which are incorporated herein byreference. In certain instances, flow cytometry systems of interestinclude BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flowcytometer, BD Biosciences FACSCelesta™ flow cytometer, BD BiosciencesFACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BDBiosciences FACSymphony™ flow cytometer BD Biosciences LSRFortessa™ flowcytometer, BD Biosciences LSRFortess™ X-20 flow cytometer and BDBiosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cellsorter, BD Biosciences FACSLyric™ cell sorter and BD Biosciences Via™cell sorter BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™cell sorter, BD Biosciences Aria™ cell sorters and BD BiosciencesFACSMelody™ cell sorter, or the like.

In some embodiments, the subject particle sorting systems are flowcytometric systems, such those described in U.S. Pat. Nos. 9,952,076;9,933,341; 9,784,661; 9,726,527; 9,453,789; 9,200,334; 9,097,640;9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300;7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804;6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; thedisclosure of which are herein incorporated by reference in theirentirety.

In certain instances, the subject systems are flow cytometry systemsconfigured for imaging particles in a flow stream by fluorescenceimaging using radiofrequency tagged emission (FIRE), such as thosedescribed in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013)as well as described in U.S. Pat. Nos. 9,423,353; 9,784,661 and10,006,852 and U.S. Patent Publication Nos. 2017/0133857 and2017/0350803, the disclosures of which are herein incorporated byreference.

Methods for Measuring Light Collected from an Irradiated Sample

Aspects of the disclosure also include methods for measuring light froma sample (e.g., in the flow stream in a flow cytometer). In practicingmethods according to embodiments, a sample is irradiated with a lightsource and light from the sample is detected with the light detectionsystems having three or more wavelength separators that are eachconfigured to pass light having a predetermined spectral range and oneor more light detection modules in optical communication with eachwavelength separator having a plurality of photodetectors and an opticalcomponent that conveys light having a predetermined sub-spectral rangeto the photodetectors as described above. In some embodiments, thesample is a biological sample. The term “biological sample” is used inits conventional sense to refer to a whole organism, plant, fungi or asubset of animal tissues, cells or component parts which may in certaininstances be found in blood, mucus, lymphatic fluid, synovial fluid,cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid,amniotic cord blood, urine, vaginal fluid and semen. As such, a“biological sample” refers to both the native organism or a subset ofits tissues as well as to a homogenate, lysate or extract prepared fromthe organism or a subset of its tissues, including but not limited to,for example, plasma, serum, spinal fluid, lymph fluid, sections of theskin, respiratory, gastrointestinal, cardiovascular, and genitourinarytracts, tears, saliva, milk, blood cells, tumors, organs. Biologicalsamples may be any type of organismic tissue, including both healthy anddiseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certainembodiments, the biological sample is a liquid sample, such as blood orderivative thereof, e.g., plasma, tears, urine, semen, etc., where insome instances the sample is a blood sample, including whole blood, suchas blood obtained from venipuncture or fingerstick (where the blood mayor may not be combined with any reagents prior to assay, such aspreservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or“mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present invention may be applied to samples from a human subject, itis to be understood that the methods may also be carried-out on samplesfrom other animal subjects (that is, in “non-human subjects”) such as,but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

In practicing the subject methods, a sample (e.g., in a flow stream of aflow cytometer) is irradiated with light from a light source. In someembodiments, the light source is a broadband light source, emittinglight having a broad range of wavelengths, such as for example, spanning50 nm or more, such as 100 nm or more, such as 150 nm or more, such as200 nm or more, such as 250 nm or more, such as 300 nm or more, such as350 nm or more, such as 400 nm or more and including spanning 500 nm ormore. For example, one suitable broadband light source emits lighthaving wavelengths from 200 nm to 1500 nm. Another example of a suitablebroadband light source includes a light source that emits light havingwavelengths from 400 nm to 1000 nm. Where methods include irradiatingwith a broadband light source, broadband light source protocols ofinterest may include, but are not limited to, a halogen lamp, deuteriumarc lamp, xenon arc lamp, stabilized fiber-coupled broadband lightsource, a broadband LED with continuous spectrum, superluminescentemitting diode, semiconductor light emitting diode, wide spectrum LEDwhite light source, an multi-LED integrated white light source, amongother broadband light sources or any combination thereof.

In other embodiments, methods includes irradiating with a narrow bandlight source emitting a particular wavelength or a narrow range ofwavelengths, such as for example with a light source which emits lightin a narrow range of wavelengths like a range of 50 nm or less, such as40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nmor less, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Where methodsinclude irradiating with a narrow band light source, narrow band lightsource protocols of interest may include, but are not limited to, anarrow wavelength LED, laser diode or a broadband light source coupledto one or more optical bandpass filters, diffraction gratings,monochromators or any combination thereof.

In certain embodiments, methods include irradiating the sample with oneor more lasers. As discussed above, the type and number of lasers willvary depending on the sample as well as desired light collected and maybe a gas laser, such as a helium-neon laser, argon laser, krypton laser,xenon laser, nitrogen laser, CO₂ laser, CO laser, argon-fluorine (ArF)excimer laser, krypton-fluorine (KrF) excimer laser, xenon chlorine(XeCl) excimer laser or xenon-fluorine (XeF) excimer laser or acombination thereof. In others instances, the methods includeirradiating the flow stream with a dye laser, such as a stilbene,coumarin or rhodamine laser. In yet other instances, methods includeirradiating the flow stream with a metal-vapor laser, such as ahelium-cadmium (HeCd) laser, helium-mercury (HeHg) laser,helium-selenium (HeSe) laser, helium-silver (HeAg) laser, strontiumlaser, neon-copper (NeCu) laser, copper laser or gold laser andcombinations thereof. In still other instances, methods includeirradiating the flow stream with a solid-state laser, such as a rubylaser, an Nd:YAG laser, NdCrYAG laser, Er:YAG laser, Nd:YLF laser,Nd:YVO₄ laser, Nd:YCa₄O(BO₃)₃ laser, Nd:YCOB laser, titanium sapphirelaser, thulim YAG laser, ytterbium YAG laser, ytterbium₂O₃ laser orcerium doped lasers and combinations thereof.

The sample may be irradiated with one or more of the above mentionedlight sources, such as 2 or more light sources, such as 3 or more lightsources, such as 4 or more light sources, such as 5 or more lightsources and including 10 or more light sources. The light source mayinclude any combination of types of light sources. For example, in someembodiments, the methods include irradiating the sample in the flowstream with an array of lasers, such as an array having one or more gaslasers, one or more dye lasers and one or more solid-state lasers.

The sample may be irradiated with wavelengths ranging from 200 nm to1500 nm, such as from 250 nm to 1250 nm, such as from 300 nm to 1000 nm,such as from 350 nm to 900 nm and including from 400 nm to 800 nm. Forexample, where the light source is a broadband light source, the samplemay be irradiated with wavelengths from 200 nm to 900 nm. In otherinstances, where the light source includes a plurality of narrow bandlight sources, the sample may be irradiated with specific wavelengths inthe range from 200 nm to 900 nm. For example, the light source may beplurality of narrow band LEDs (1 nm-25 nm) each independently emittinglight having a range of wavelengths between 200 nm to 900 nm. In otherembodiments, the narrow band light source includes one or more lasers(such as a laser array) and the sample is irradiated with specificwavelengths ranging from 200 nm to 700 nm, such as with a laser arrayhaving gas lasers, excimer lasers, dye lasers, metal vapor lasers andsolid-state laser as described above.

Where more than one light source is employed, the sample may beirradiated with the light sources simultaneously or sequentially, or acombination thereof. For example, the sample may be simultaneouslyirradiated with each of the light sources. In other embodiments, theflow stream is sequentially irradiated with each of the light sources.Where more than one light source is employed to irradiate the samplesequentially, the time each light source irradiates the sample mayindependently be 0.001 microseconds or more, such as 0.01 microsecondsor more, such as 0.1 microseconds or more, such as 1 microsecond ormore, such as 5 microseconds or more, such as 10 microseconds or more,such as 30 microseconds or more and including 60 microseconds or more.For example, methods may include irradiating the sample with the lightsource (e.g. laser) for a duration which ranges from 0.001 microsecondsto 100 microseconds, such as from 0.01 microseconds to 75 microseconds,such as from 0.1 microseconds to 50 microseconds, such as from 1microsecond to 25 microseconds and including from 5 microseconds to 10microseconds. In embodiments where sample is sequentially irradiatedwith two or more light sources, the duration sample is irradiated byeach light source may be the same or different.

The time period between irradiation by each light source may also vary,as desired, being separated independently by a delay of 0.001microseconds or more, such as 0.01 microseconds or more, such as 0.1microseconds or more, such as 1 microsecond or more, such as 5microseconds or more, such as by 10 microseconds or more, such as by 15microseconds or more, such as by 30 microseconds or more and includingby 60 microseconds or more. For example, the time period betweenirradiation by each light source may range from 0.001 microseconds to 60microseconds, such as from 0.01 microseconds to 50 microseconds, such asfrom 0.1 microseconds to 35 microseconds, such as from 1 microsecond to25 microseconds and including from 5 microseconds to 10 microseconds. Incertain embodiments, the time period between irradiation by each lightsource is 10 microseconds. In embodiments where sample is sequentiallyirradiated by more than two (i.e., 3 or more) light sources, the delaybetween irradiation by each light source may be the same or different.

The sample may be irradiated continuously or in discrete intervals. Insome instances, methods include irradiating the sample in the samplewith the light source continuously. In other instances, the sample in isirradiated with the light source in discrete intervals, such asirradiating every 0.001 millisecond, every 0.01 millisecond, every 0.1millisecond, every 1 millisecond, every 10 milliseconds, every 100milliseconds and including every 1000 milliseconds, or some otherinterval.

Depending on the light source, the sample may be irradiated from adistance which varies such as 0.01 mm or more, such as 0.05 mm or more,such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more,such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more,such as 15 mm or more, such as 25 mm or more and including 50 mm ormore. Also, the angle or irradiation may also vary, ranging from 10° to90°, such as from 15° to 85°, such as from 20° to 80°, such as from 25°to 75° and including from 30° to 60°, for example at a 90° angle.

As discussed above, in embodiments light from the irradiated sample isconveyed to a light detection system as described herein and measured byone or more photodetectors. In practicing the subject methods, lightfrom the sample is conveyed to three or more wavelength separators thatare each configured to pass light having a predetermined spectral range.The spectral ranges of light from each of the wavelength separators areconveyed to one or more light detection modules having opticalcomponents that are configured to convey light having a predeterminedsub-spectral range to the photodetectors.

Light may be measured with the light detection systems continuously orin discrete intervals. In some instances, methods include takingmeasurements of the light continuously. In other instances, the light ismeasured in discrete intervals, such as measuring light every 0.001millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1millisecond, every 10 milliseconds, every 100 milliseconds and includingevery 1000 milliseconds, or some other interval.

Measurements of the collected light may be taken one or more timesduring the subject methods, such as 2 or more times, such as 3 or moretimes, such as 5 or more times and including 10 or more times. Incertain embodiments, the light propagation is measured 2 or more times,with the data in certain instances being averaged.

In some embodiments, methods include adjusting the light beforedetecting the light with the subject light detection systems. Forexample, the light from the sample source may be passed through one ormore lenses, mirrors, pinholes, slits, gratings, light refractors, andany combination thereof. In some instances, the collected light ispassed through one or more focusing lenses, such as to reduce theprofile of the light directed to the light detection system or opticalcollection system as described above. In other instances, the emittedlight from the sample is passed through one or more collimators toreduce light beam divergence conveyed to the light detection system.

Kits

Aspects of the invention further include kits, where kits include threeor more wavelength separators, a plurality of photodetectors and one ormore optical components (e.g., dichroic mirrors, beam splitters,collimating lenses, etc.). In some embodiments, kits include a substratefor co-mounting a wavelength separator with an optical component and aphotodetector. In certain embodiments, kits include one or morefasteners for assembling together components of the subject lightdetection systems. Kits may also include an optical collectioncomponent, such as fiber optics (e.g., fiber optics relay bundle) orcomponents for a free-space relay system. In some instances, kitsfurther include one or more photodetectors, such as photomultipliertubes (e.g., metal package photomultiplier tubes).

In some embodiments, kits include 2 or more of the components of thelight detection systems disclosed herein, such as 3 or more andincluding 5 or more. In some instances, the kits can include one or moreassay components (e.g., labeled reagents, buffers, etc., such asdescribed above). In some instances, the kits may further include asample collection device, e.g., a lance or needle configured to prickskin to obtain a whole blood sample, a pipette, etc., as desired.

The various assay components of the kits may be present in separatecontainers, or some or all of them may be pre-combined. For example, insome instances, one or more components of the kit are present in asealed pouch, e.g., a sterile foil pouch or envelope.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), portable flash drive, and the like, on which the information hasbeen recorded. Yet another form of these instructions that may bepresent is a website address which may be used via the internet toaccess the information at a removed site.

Utility

The subject light detection systems find use where the characterizationof a sample by optical properties, in particular where low levels oflight are collected, is desired. In some embodiments, the systems andmethods described herein find use in flow cytometry characterization ofbiological samples labelled with fluorescent tags. In other embodiments,the systems and methods find use in spectroscopy of transmitted orscattered light. In addition, the subject systems and methods find usein increasing the obtainable signal from light collected from a sample(e.g., in a flow stream). In certain instances, the present disclosurefinds use in enhancing measurement of light collected from a sample thatis irradiated in a flow stream in a flow cytometer. Embodiments of thepresent disclosure find use where enhancing the effectiveness ofemission measurements in flow cytometry are desired, such as in researchand high throughput laboratory testing. The present disclosure alsofinds use where it is desirable to provide a flow cytometer withimproved cell sorting accuracy, enhanced particle collection, reducedenergy consumption, particle charging efficiency, more accurate particlecharging and enhanced particle deflection during cell sorting.

The present disclosure also finds use in applications where cellsprepared from a biological sample may be desired for research,laboratory testing or for use in therapy. In some embodiments, thesubject methods and devices may facilitate the obtaining of individualcells prepared from a target fluidic or tissue biological sample. Forexample, the subject methods and systems facilitate obtaining cells fromfluidic or tissue samples to be used as a research or diagnosticspecimen for diseases such as cancer. Likewise, the subject methods andsystems facilitate obtaining cells from fluidic or tissue samples to beused in therapy. Methods and devices of the present disclosure allow forseparating and collecting cells from a biological sample (e.g., organ,tissue, tissue fragment, fluid) with enhanced efficiency and low cost ascompared to traditional flow cytometry systems.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

1.-139. (canceled)
 140. A method comprising: detecting light from theflow stream with a light detection system comprising: three or morewavelength separators that are each configured to pass light having apredetermined spectral range; and one or more light detection modules inoptical communication with each wavelength separator, wherein each lightdetection module comprises: a plurality of photodetectors; and anoptical component configured to convey light having a predeterminedsub-spectral range to the photodetectors.
 141. The method according toclaim 140, further comprising irradiating a sample in a flow stream inan interrogation field with a light source.
 142. The method according toclaim 140, wherein the flow stream is irradiated with a light source ata wavelength from 200 nm to 800 nm.
 143. The method according to claim141, wherein the light source is a laser.
 144. The method according toclaim 141, wherein light from the flow stream is conveyed to the lightdetection system with an optical collection component.
 145. The methodaccording to claim 144, wherein the optical collection componentcomprises fiber optics.
 146. The method according to claim 145, whereinthe optical collection component comprises a fiber optics light relaybundle.
 147. The method according to claim 144, wherein the opticalcollection component comprises a free-space light relay system.
 148. Amethod comprising: detecting light from the flow stream with a lightdetection system comprising: a wavelength separator configured togenerate first, second and third predetermined spectral ranges of lightfrom a light source; and first, second and third light detection modulesconfigured to receive each of the first, second and third predeterminedspectral ranges of light, wherein each of the first, second and thirdlight detection modules comprises: a plurality of photodetectors; and anoptical component configured to convey light having a predeterminedsub-spectral range to the photodetectors.
 149. The method according toclaim 148, further comprising irradiating a sample in a flow stream inan interrogation field with a light source.
 150. The method according toclaim 148, wherein the flow stream is irradiated with a light source ata wavelength from 200 nm to 800 nm.
 151. The method according to claim149, wherein the light source is a laser.
 152. The method according toclaim 148, wherein light from the flow stream is conveyed to the lightdetection system with an optical collection component.
 153. The methodaccording to claim 152, wherein the optical collection componentcomprises fiber optics.
 154. The method according to claim 153, whereinthe optical collection component comprises a fiber optics light relaybundle.
 155. The method according to claim 152, wherein the opticalcollection component comprises a free-space light relay system.
 156. Amethod comprising: detecting light from the flow stream with a lightdetection system comprising: a wavelength separator configured togenerate first, second and third predetermined spectral ranges of lightfrom a light source; and first, second and third light detection modulesconfigured to receive each of the first, second and third predeterminedspectral ranges of light, wherein each of the first, second and thirdlight detection modules are configured to generate a plurality ofsub-spectral ranges of light, wherein each sub-spectral range of lightexhibits a light loss of 20% or less as compared to the light from thelight source.
 157. The method according to claim 156, wherein eachsub-spectral range of light exhibits a light loss of 15% or less ascompared to the light from the light source.
 158. The method accordingto claim 156, wherein the first, second and third light detectionmodules are configured to generate 20 or more sub-spectral ranges oflight.
 159. The method according to claim 156, wherein the first, secondand third light detection modules each comprise a plurality of dichroicmirrors and are configured to generate the 20 or more sub-spectralranges of light with 10 reflections or less.
 160. The method accordingto claim 156, further comprising irradiating a sample in a flow streamin an interrogation field with a light source.
 161. The method accordingto claim 156, wherein the flow stream is irradiated with a light sourceat a wavelength from 200 nm to 800 nm.
 162. The method according toclaim 160, wherein the light source is a laser.
 163. The methodaccording to claim 156, wherein light from the flow stream is conveyedto the light detection system with an optical collection component. 164.The method according to claim 163, wherein the optical collectioncomponent comprises fiber optics.
 165. The method according to claim163, wherein the optical collection component comprises a fiber opticslight relay bundle.
 166. The method according to claim 163, wherein theoptical collection component comprises a free-space light relay system.167.-175. (canceled)